For the medicinal treatment of gallstone problems, the bile salt active substances ursodesoxycholic acid (UDCS or UDCA) and the corresponding diastereomer chenodesoxycholic acid (CDCS or CDCA) inter alia have been used for many years. The two compounds differ only in the configuration of the hydroxy group at C atom 7 (UDCA: β-configuration, CDCA: α-configuration). In the prior art, various methods are described for the production of UDCA, which are performed purely chemically or consist of a combination of chemical and enzymatic process steps. The starting point in each case is cholic acid (CA or CA) or CDCA produced from cholic acid.
Thus the classical chemical method for UDCA production can be represented schematically as follows:

Inter alia, a serious disadvantage is as follows: since the chemical oxidation is not selective, the carboxy group and the 3α and 7α-hydroxy group must be protected by esterification.
An alternative chemical/enzymatic method based on the use of the enzyme 12α-hydroxysteroid dehydrogenase (12α-HSDH) can be represented as follows and is for example described in PCT/EP2009/002190 by the present applicant.

Here the 12α-HSDH oxidizes CA selectively to 12-keto-CDCA. As a result, the two protection steps necessary according to the classical chemical methods become superfluous.
Furthermore, an alternative enzymatic/chemical method is described by Monti, D., et al., (One-Pot Multienzymatic Synthesis of 12-ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row. Advanced Synthesis & Catalysis, 2009), which is schematically representable as follows:

The CA is firstly oxidized to 7,12-diketo-LCA by 7α-HSDH from Bacteroides fragilis ATCC 25285 (Zhu, D., et al., Enzymatic enantioselective reduction of keto esters by a thermostable 7-hydroxysteroid dehydrogenase from Bacteroides fragilis. Tetrahedron, 2006. 62(18): p. 4535-4539) and 12α-HSDH. These two enzymes are both NADH-dependent. After the reduction by 7β-HSDH (NADPH-dependent) from Clostridium absonum ATCC 27555 (DSM 599) (MacDonald, I. A. and P. D. Roach, Bile induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium absonum. Biochim Biophys Acta, 1981. 665(2): p. 262-9), 12-keto-UDCA is formed. The end product is obtained by Wolff-Kishner reduction. In this method, it is disadvantageous that because of the equilibrium position of the catalyzed reaction a complete conversion is not possible, and that for the first stage of the conversion two different enzymes must be used, which renders the method expensive. For the cofactor regeneration, lactate dehydrogenase (LDH; for regeneration of NAD+) and glucose dehydrogenase (GlcDH or GDH, for regeneration of NADPH) are used. In the cofactor regeneration used there, it is disadvantageous that the co-product produced can only be removed from the reaction mixture with great difficulty, so that the reaction equilibrium cannot be favorably influenced, which results in incomplete conversion of the educt.
A 7β-HSDH from the strain Collinsella aerofaciens ATCC 25986 (DSM 3979; former Eubacterium aerofaciens) was described in 1982 by Hirano and Masuda (Hirano, S. and N. Masuda, Characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofaciens. Appl Environ Microbiol, 1982. 43(5): p. 1057-63). Sequence information on this enzyme was not disclosed. The molecular weight determined by gel filtration was 45,000 Da (see Hirano, page 1059, left-hand column). Furthermore, the reduction of the 7-oxo group to the 7β-hydroxy group could not be observed for the enzyme there (see Hirano, page 1061, Discussion 1stparagraph). Those skilled in the art thus recognize that the enzyme described by Hirano et al is not suitable for the catalysis of the reduction of dehydrocholic acid (DHCA or DHCA) in the 7-position to 3,12-diketo-UDCA.
In the applicant's WO2011/064404, a novel 7β-HSDH from Collinsella aerofaciens ATCC 25986 is described, which inter alia has a molecular weight (on SDS gel electrophoresis) of about 28-32 kDa, a molecular weight (on gel filtration, under non-denaturing conditions, such as in particular without SDS) of about 53 to 60 kDa, and has the capacity to effect the stereoselective reduction of the 7-carbonyl group of 7-keto LCA to a 7β-hydroxy group.
Apart from this, in WO2011/064404 a method for UDCA production is provided, which is schematically representable as follows:

Here the oxidation of CA takes place simply by a classical chemical route. The DHCA is reduced to 12-keto-UDCA by the enzyme pair 7β-HSDH and 3α-HSDH singly in succession or in one pot. Combined with the Wolff-Kishner reduction, UDCA can thus be synthesized from CA in only three steps. While the 7β-HSDH is dependent on the cofactor NADPH, the 3α-HSDH requires the cofactor NADH. The availability of enzyme pairs with dependence on the same cofactor or extended dependence (e.g. on the cofactors NADH and NADPH) would be advantageous, since the cofactor regeneration could thereby be simplified
In the applicant's WO2012/080504, in particular a whole cell method for biocatalytic reduction of dehydrocholic acid compounds (DHCA) and in particular a novel method for producing UDCA using recombinant whole cell catalysts which express 7β-HSDH and 3α-HSDH and wherein the enzymatic reduction steps for the cofactor regeneration are coupled with a cofactor-regenerating enzyme, such as for example a suitable glucose dehydrogenase (GDH), is described.
Critical for the efficiency of an enzymatic synthesis is the manner in which the required enzymes are used. In this, whole cell biocatalysis is a proven approach. Here, the often heterologous enzymes are overexpressed within the host organism and the cell as a whole is used as a biocatalyst. A special whole cell biocatalyst from WO2012/080504 heterologously expresses a for example NADPH-dependent, 7β-HSDH from Collinsella aerofaciens, a for example NADH-dependent 3α-HSDH from Comamonas testosteroni and a GDH utilizing both NADH and also NADPH from Bacillus subtilis and is used as a whole cell biocatalyst for the reduction of DHCA to 12-keto-ursodesoxycholic acid (12-keto-UDCA). There, for example 17.7 gBDM L−1 biocatalyst were used to convert 100 mM DHCA 98% to 12-keto-UDCA. Since biocatalysts are a main cost factor in this production process, the present technical challenge consists in the discovery of technical solutions in which the process costs can be decreased, for example by partially replacing the biocatalysts by other substances.
A first objective therefore consists in the provision of novel biocatalytic processes which are characterized in particular by higher cost efficiency in the reductive production of UDCA via DHCA.
A further objective of the invention is the provision of further improved 7β-HSDHs. In particular, enzyme mutants should be provided which can still more advantageously be used for the enzymatic or microbial (biocatalytic) production of UDCA via the stereospecific reduction of DHCA in the 7-position, and in particular have a modified cofactor usage (in particular an improved NADH specificity).